The objective of the proposed research is to continue work related to characterization of the transporter protein for long chain fatty acids. We have synthesized sulfosuccinimidyl derivatives of long-chain fatty acids and investigated their effect on fatty acid transport and their binding to adipocyte membrane proteins. The oleate derivative inhibited transport and specifically labeled a membrane protein of about 88 kd. This protein was common to three irreversible inhibitors of fatty acid transport and thus a strong candidate for the fatty acid transporter. The protein was isolated and its amino-terminal sequence was determined. Using an oligonucleotide derived from the N-terminal sequence, we have isolated a clone from a rat fat cDNA library which has the entire coding sequence. We propose to express this clone in a suitable recipient cell such as xenopus oocytes or mammalian cells in order to establish the role of the protein in FA transport. Structurefunction studies will be carried out by deletion or addition of nucleotide sequences and by site-directed mutagenesis coupled with functional expression or with reconstitution into liposomes. We will determine protein domains which are important for fatty acid binding and transport and explore whether protein activity is regulated by phosphorylation with kinases. Long-term goals include determination of tissue distribution, possible existence of isoforms, and regulation of protein levels by hormones or disease states. Fatty acids are the major energy substrate in humans and essential precursors of membrane phospholipids. They are also modulators or mediators of membrane channels, receptors and enzymes so their role in regulating multiple aspects of cell function is potentially large. Understanding the molecular mechanism which mediates cellular uptake of long chain fatty acids is undoubtedly important for the general physiology of the organism in health and disease.